Work supported by this grant over the years helped to develop the hydrogen exchange knowledge and methods that are now widely exploited in studies of protein structure and function. Progress during this grant period has uncovered a fundamental but previously unsuspected protein property. It appears that proteins are composed of a small number of coooperative unfolding/refolding units, called foldons, and that these units form and assemble in a pathway sequence through distinct intermediate forms to progressively construct the native protein. The present application proposes to further test this hypothesis in cytochrome c. Also the generality of this behavor will be tested with other major protein models, using staphylococcal nuclease initially and then other model proteins to be selected according to specified criteria. In other work to continue the development of hydrogen exchange knowledge and its use for understanding protein dynamics and signal transfer, designed mutational perturbations will be used to study the local fluctuation mechanisms of proteins and the internal protein pathways that mediate more distant changes. All of this work will use special hydrogen exchange methods that we have developed and will depend on NMR analysis. Related hydrogen exchange experiments that depend on mass spectroscopic analysis, together with other methods, will be used to study amyloid structures and their precursor forms. The focus will be on a-synuclein initially (Parkinson's Disease) and then A|3 (Alzheimer's Disease) and will use high throughput methods among others, looking toward drug discovery efforts. The research described here aims to understand the functioning of the protein machinery of biology, both in health and in disease. This work has uncovered and is continuing to study a previously unsuspected property of protein molecules, their cooperative foldon substructure, which appears to explain how proteins fold to their active form and how they perform some other functions. The knowledge gained and the special methods developed are also being used to study the protein misfolding mechanisms that lead to devastating human and animal neurodegenerative pathologies such as Parkinson's and Alzheimer's disease and bovine spongiform encephalopathy (mad cow disease).